First, canines (n=24) with Staphylococcus aureus (S. aureus) pneumonia were randomized in a blinded fashion for exchange-transfusion with either 7- or 42-day-old canine commercially available universal donor blood (80 mL/kg in 4 divided doses). These experiments were designed to test the extremes of storage age and transfusion volume. Older blood transfusion increased mortality, the arterial-alveolar oxygen gradient and histological lung damage. Older blood resulted in increased in vivo hemolysis, releasing free iron (in the form of non-transferrin bound iron) and CFH and decreasing haptoglobin plasma levels. Consistent with the vasoconstrictive effect of CFH, older blood increased both systemic and pulmonary pressures. This was the first randomized blinded animal trial showing blood transfused at end of storage period can increase mortality during infection. Next, we investigated the effect of increasing bacterial doses and severity of infection on the risks associated with age of blood transfused in this canine model. A range of S. aureus bacterial doses were administered and then exchange-transfused with either 7- or 42-day-old canine universal donor blood (80 mL/kg in four divided doses). Without bacterial challenge, levels of CFH and NTBI were significantly higher with older versus fresher blood transfusion but there were no significant differences in measurable injury. With higher-dose bacterial challenge, the elevated NTBI levels declined more rapidly and to a greater extent after transfusion with older versus fresher blood, and older blood was associated with significantly worse shock, lung injury, and mortality. The CFH levels were markedly elevated over days regardless of severity of infection. The augmented in vivo hemolysis of transfused older RBCs, resulting in excess plasma CFH and iron release, appears to require the presence of established infection to worsen outcomes. These data suggest that transfused older RBCs increases the risks specifically from infection in septic subjects and define an infection dose-response. During canine bacterial pneumonia with septic shock, but not in controls, older stored RBCs were associated with significantly increased lung injury and mortality. We wondered whether transfusion of older RBCs would cause similar adverse effects during shock and inflammatory injury without infection. Therefore, animals (n=12) were transfused similar quantities of either older (42-day) or fresher (7-day) stored universal donor canine RBCs 2.5 hours after undergoing controlled hemorrhage producing shock. With older transfused RBCs, CFH and NTBI levels increased, but lung injury declined and there was a trend toward lower mortality (18% vs. 50%). Interestingly, the increased levels of CFH with older blood transfusion were associated with an improved hemodynamic response to hemorrhage-reperfusion, with lowered exogenous norepinephrine requirements and cardiac outputs. This hemodynamic effect is consistent with the ability of CFH to scavenge NO causing vasoconstriction. Thus, in hemorrhagic shock, older RBCs altered resuscitation physiology but did not worsen clinical outcomes. Elevated CFH lowers norepinephrine requirements and cardiac output, ameliorating reperfusion injury. In our infection model, we had previously shown that older blood increases NTBI levels transiently during transfusion and the rapid clearance of iron is associated with increased lung injury and mortality. In contrast, during hemorrhagic shock, NTBI levels persist longer after transfusion, and these persistently increased levels of iron are not associated with worsened outcomes. These preclinical data suggest that, whereas iron derived from older RBCs promotes bacterial growth worsening septic shock mortality during infection, release of CFH and NTBI during hemorrhagic shock is not necessarily harmful. We next conducted a blinded randomized controlled study of RBC washing in this canine model of transfusion injury. We hypothesized that washing older units of blood before transfusion would improve clinical outcomes by removing older fragile RBCs and prevent increases of CFH and iron, whereas washing fresher units would have no effect on outcome. Twenty-four animals with S. aureus pneumonia, were exchange-transfused with either 7- or 42-day-old washed (commercially available Haemonetics blood cell processor with standard washing procedure) or unwashed canine universal donor blood (80 mL/kg in 4 divided doses). Washing older units of blood improved survival rates, shock score, lung injury, cardiac performance and liver function, and reduced levels of NTBI, possibly by lysing and washing away older cells and supernatant. In contrast, washing fresh blood worsened all these same clinical parameters and increased CFH levels. Our data suggest that fresh blood should not be washed routinely because washing induces sub-lethal membrane damage to the RBC and, in a setting of established infection, washed RBCs are prone to lyse, release CFH, and result in worsened clinical outcomes. These findings, along with our previous studies, indicate that transfusion of fresh blood in infected subjects results in less hemolysis, CFH, and iron release, and is less toxic than transfusion of older blood in critically ill infected subjects. However, if older blood must be used during established infection, washing prevents elevations in plasma circulating iron and improves survival and lessens multiple organ injury. We next examined the results of altering volume, washing, and age of RBCs. Animals were transfused with increasing volumes (5-10, 20-40, or 60-80 mL/kg) of either 42- or 7-day-old RBCs (n=36) or 80 mL/kg of either unwashed or washed RBCs with increasing storage age (14, 21, 28, or 35 days) (n=40). All volumes transfused (5-80 mL/kg) of 42-day-old RBCs resulted in like (i.e., not significantly different) increases in iron, CFH, lung injury, and mortality rates after transfusion. Transfusion of 80 mL/kg of RBCs stored for 14, 21, 28 and 35 days resulted in increased CFH and NTBI in between levels found at 7 and 42 days of storage. However, washing RBCs of intermediate ages (14-35 days) does not alter NTBI and CFH levels or mortality rates. Thus, our preclinical data suggest that any volume of 42-day-old blood potentially increases risks during established infection. In contrast, even massive volumes of 7-day-old blood result in minimal CFH and NTBI levels and risks. In contrast to the extremes of storage, washing blood stored for intermediate ages does not alter risks of transfusion or NTBI and CFH clearance.8 At present, we are investigating whether haptoglobin therapy improves outcome after blood transfusion with different RBC storage ages as well as during sepsis without transfusion. Transfusion and bacterial sepsis are both independent potential sources of elevated plasma CFH. Increased CFH levels after transfusion and during sepsis have both been associated with worse outcomes in animal models and humans. Haptoglobin binds CFH and promotes its clearance.